Recombinant human serum albumin, process for producing the same and pharmaceutical preparation containing the same

ABSTRACT

Human serum albumin obtained by gene manipulation techniques can be purified by a combination of specified steps in which a culture supernatant obtained from a human serum albumin-producing host is subjected to ultrafiltration, heat treatment, acid treatment and another ultrafiltration, followed by subsequent treatments with a cation exchanger, a hydrophobic chromatography carrier and an anion exchanger, and by salting-out to thereby obtain a pure form of human serum albumin which contains substantially no proteinous and polysaccharide contaminants, which is formulated into a pharmaceutical preparation. The thus obtained human serum albumin can further be purified by treating recombinant human serum albumin with a hydrophobic chromatography carrier at pH of 2 to 5 and a salt concentration of 0.4 to 1 and exposing the carrier to a pH of 6 to 8 and a salt concentration of 0.01 to 0.3 M, or treating the culture supernatant with boric acid or a salt thereof at pH 8 to 11 for 1 to 10 hours and recovering the supernatant. This process makes it possible to effeciently purify recombinant human serum albumin and to provide substantially pure human serum albumin which does not contain producer host-related substances and other contaminants and is sufficiently free from coloration.

CROSS-REFERENCES TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/202,130 filed Feb. 25,1994 now U.S. Pat. No. 5,521,287 which is a continuation-in-part ofapplication Ser. No. 08/036,387 filed on Mar. 24, 1993 now U.S. Pat. No.5,440,018.

FIELD OF THE INVENTION

The instant invention relates to a process for producing recombinanthuman serum albumin, in which the human serum albumin is purifiedthrough a combination of steps to yield a substantially pure form ofhuman serum albumin.

BACKGROUND OF THE INVENTION

Albumin, especially human serum albumin (HSA), is an important proteinof the circulatory system. The protein is produced in the liver and hasa major role in maintaining normal osmotic pressure of body fluids, suchas blood. It also serves as a carrier of various molecules.

HSA is administered under various clinical conditions. For example, inthe case of shock or burn injury, it is necessary, in general, toadminister HSA frequently to restore blood volume and to alleviate otherinjury-related symptoms. Patients suffering from hypoproteinemia andfetal erythroblastosis sometimes require HSA treatment.

In other words, a common indication for HSA administration is a loss ofbody fluids, such as during a surgical procedure, shock, burn injury orhypoproteinemia which causes edema.

Currently, HSA is produced mainly as a fractionated product of collectedblood. Such a production process, however, has disadvantages in that itis not economical and the supply of blood is sporadic. In addition,collected blood sometimes contains undesirable substances, such ashepatitis virus. In consequence, it is profitable to develop a materialwhich can be used as an HSA substitute.

Recent advances in recombinant DNA techniques have rendered possiblemicrobial production of various types of useful polypeptides, and, as aresult, a number of mammalian polypeptides already have been produced invarious microorganisms. With regard to HSA, establishing techniques forthe large scale production of HSA by recombinant methods and subsequenthigh grade purification also is in progress.

Techniques for the isolation and purification of HSA from plasma havebeen studied from various points of view and put into practical use. Forexample, the ethanol fractionation method of E. J. Cohn et al., PEGfractionation method, ammonium sulfate fractionation method and the likeare well known methods. In addition to those methods, severalpurification processes recently have been developed, such as, forexample, a process in which an anion exchanger treatment and a heattreatment at 60° C. for 10 hours are employed in combination(JP-A-2-191226 corresponding to EP-A-367220) and a process in which ananion exchanger treatment, a cation exchanger treatment and a heattreatment at 60° C. for 10 hours are employed in combination(JP-A-3-17123 corresponding to EP-A-428758). (The term "JP-A" as usedherein means an "unexamined published Japanese patent application".)

However, in the case of producing HSA by means of gene manipulationtechniques, it is highly probable that an HSA preparation of interestwill be contaminated by certain coloring components which are containedin the raw materials or secreted by a microorganism during culturing ofthe host microorganism or are introduced during purification of theresulting HSA, and that those contaminants bind to HSA to cause coloringof the HSA itself. What is more, such contaminants cannot be removedsufficiently by means of any prior art process for the purification ofplasma-derived HSA.

SUMMARY OF THE INVENTION

Taking the aforementioned problems involved in the prior art intoconsideration, the instant inventors have conducted intensive studiesand, as a result, succeeded in developing a process for efficientlypurifying HSA obtained by gene manipulation techniques.

An object of the instant invention is to provide human serum albuminobtained by means of gene manipulation techniques, which does notcontain producer host-related substances or other contaminants and issubstantially free from coloring.

More specifically, the instant invention provides a process forproducing a recombinant human serum albumin comprising the steps of:

(1) treating a culture supernatant of a host which expresses human serumalbumin, with a first ultrafiltration membrane having a molecular weightexclusive limit of from 100,000 to 500,000 and then with a secondultrafiltration membrane having a molecular weight exclusive limit offrom 1,000 to 50,000 to yield a first filtrate;

(2) heat-treating the first filtrate at 50 to 70° C. for 30 minutes to 5hours to yield a heated sample;

(3) acid-treating the heated sample at a pH of 3 to 5 to yield anacid-treated sample;

(4) treating the acid-treated sample using an ultrafiltration membranehaving a molecular weight exclusive limit of from 100,000 to 500,000 toyield a second filtrate;

(5) allowing the second filtrate to contact with a cation exchanger at apH of 3 to 5 and a salt concentration of 0.01 to 0.2 M, and thenexposing the cation exchanger to a pH of 8 to 10 and a saltconcentration of 0.2 to 0.5 M to yield a first eluate;

(6) allowing the first eluate to contact with a carrier for hydrophobicchromatography at a pH of 6 to 8 and a salt concentration of 0.01 to 0.5M, and recovering non-adsorbed fractions to yield a second eluate; and

(7) allowing the second eluate to contact with an anion exchanger at apH of 6 to 8 and recovering non-adsorbed fractions to yield the albumin.

An additional step (8) may be employed in the above process, in whichthe resulting eluate of step (7) further is allowed to contact with achelate resin and the resulting non-adsorbed fractions are recovered.

Another object of the instant invention is to provide a process forproducing a recombinant human serum albumin which substantially containno free nonantigenic contaminants detectable by the phenol-sulfuric acidmethod, which comprises the steps of allowing the recombinant humanserum albumin to contact with a carrier for hydrophobic chromatographyat a pH of 2 to 5 and a salt concentration of 0.4 to 1 M and exposingthe carrier to a pH of 6 to 8 and a salt concentration of 0.01 to 0.3 M,or treating the recombinant human serum albumin with boric acid or asalt thereof to remove contaminants, which may be followed by treatmentwith a ultrafiltration membrane having a molecular weight exclusivelimit of about 100,000.

Another object of the instant invention is to provide a substantiallypure recombinant human serum albumin, wherein a 25% solution of thealbumin contains contaminated proteins in an amount of 0.1 ng/ml or lessand contaminated polysaccharides in an amount of 1 ng/ml or less.

Another object of the instant invention is to provide a recombinanthuman serum albumin, wherein a content of free nonantigenic contaminantsdetectable by the phenol-sulfuric acid method is 1 μg or less per 250 mgof said albumin, a content of producer host-related antigeniccontaminants is 0.1 ng or less and a content of pyrogen is 0.1 EU orless.

Still another object of the instant invention is to provide apharmaceutical preparation comprising recombinant human serum albumin,acetyltryptophan or a salt thereof and sodium caprylate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the results of HPLC analysis of HSA obtainedafter the hydrophobic chromatography purification step.

FIG. 2 is a graph showing binding curves of lauric acid to HSA.

DETAILED DESCRIPTION OF THE INVENTION

The term "substantially pure HSA" used herein means that a 25 w/v %aqueous solution of purified HSA contains contaminated proteins andpolysaccharides in an amount of at most 0.1 ng/ml or below and 1 ng/mlor below, respectively, or that the purity of purified HSA is 99.999999%or more.

In the process of the instant invention, the step (6) may be replaced byanother step (6) in which the resulting eluate of the step (5) isallowed to contact with a carrier for hydrophobic chromatography at a pHof 6 to 8 and a salt concentration of 1 to 3 M, and then the carrier isexposed to a pH of 6 to 8 and a salt concentration of 0.01 to 0.5 M.

Also, the step (7) of the process of the present invention may bereplaced by another step (7) in which the resulting eluate of the step(6) is allowed to contact with an anion exchanger at a pH of 6 to 8 anda salt concentration of 0.001 to 0.05 M, and then the anion exchanger isexposed to a pH of 6 to 8 and a salt concentration of 0.05 to 1 M.

In addition, the process of the present invention may further comprise asalt precipitation (salting-out) step following step (5), step (6) orstep (7), in which the salt precipitation is carried out by exposing thefirst eluate, the second eluate or the albumin to a pH of 3 to 5 and asalt concentration of 0.5 to 3 M to yield a precipitation and dissolvingthe precipitate in a buffer.

The instant invention is described in detail below.

(1) Recombinant HSA

The HSA-producing host prepared by means of gene manipulation techniquesto be used in the instant invention is not limited so long as the HSAhas been prepared via gene manipulation techniques, hence the host canbe selected from hosts already known in the art, as well as those hostswhich will be developed in the future. Illustrative examples of the hostinclude microbial cells, such as Escherichia coli, various yeastspecies, Bacillus subtilis and the like, and animal cells. Particularlypreferred hosts are yeast species, especially those belonging to thegenus Saccharomyces, such as Saccharomyces cerevisiae, or the genusPichia, such as Pichia pastoris. Auxotrophic strains orantibiotic-sensitive strains also may be used. Saccharomyces cerevisiaeAH22 (a, his 4, leu 2, can 1), Pichia pastoris GTS115 (his 4) and thelike strains are used preferably.

Preparation of the HSA-producing hosts, production of HSA by culturingthe hosts and isolation and recovery of HSA from the resulting culturebroth may be effected using known techniques or modified proceduresthereof. For example, preparation of an HSA-producing host (or anHSA-producing strain) may be effected using a process in which a naturalhuman serum albumin gene is used (JP-A-58-56684 corresponding toEP-A-73646, JP-A-58-90515 corresponding to EP-A-79739 and JP-A-58-150517corresponding to EP-A-91527), a process in which a modified human serumalbumin gene is used (JP-A-62-29985 and JP-A-1-98486 corresponding toEP-A-206733), a process in which a synthetic signal sequence is used(JP-A-1-240191 corresponding to EP-A-329127), a process in which a serumalbumin signal sequence is used (JP-A-2-167095 corresponding toEP-A-319641), a process in which a recombinant plasmid is introducedinto chromosome (JP-A-3-72889 corresponding to EP-A-399455), a processin which hosts are fused (JP-A-3-53877 corresponding to EP-A-409156), aprocess in which mutation is generated in a methanol containing medium,a process in which a mutant AOX2 promoter is used (EP-A-506040), aprocess in which HSA is expressed in B. subtilis (JP-A-62-215393corresponding to EP-A-229712), a process in which HSA is expressed inyeast (JP-A-60-41487 corresponding to EP-A-123544, JP-A-63-39576corresponding to EP-A-248657 and JP-A-63-74493 corresponding toEP-A-251744) and a process in which HSA is expressed in Pichia(JP-A-2-104290 corresponding to EP-A-344459).

The process in which mutation is generated in a methanol-containingmedium is carried out in the following manner.

Firstly, a plasmid containing a transcription unit which is constructedso as to express HSA under the control of AOX1 promoter is introducedinto the AOX1 gene region of an appropriate host, preferably a Pichiayeast, more preferably Pichia strain GTS115 (NRRL deposition numberY-15851) (JP-A-2-104290 corresponding to EP-A-344459) to obtain atransformant. Since the thus obtained transformant does not grow well ina methanol-containing medium, mutation of the transformant is effectedby culturing the transformant in a methanol-containing medium to isolatea mutant strain which is capable of growing in the medium. The methanolconcentration in the medium may be in the range of approximately from0.01 to 5%. The medium may be either synthetic or natural, and theculturing may be carried out at 15 to 40° C. for 1 to 1,000 hours.

Culturing of an HSA-producing host (an HSA production process) may becarried out using known processes disclosed in the aforementionedreferences, or in accordance with a process disclosed in JP-A-3-83595 inwhich high concentration substrate inhibition of HSA producer cells isavoided by gradually adding a high concentration glucose solution to amedium by means of fed batch fermentation, thereby enabling productionof both the producer cells and the product in high concentrations, or inaccordance with another process disclosed in JP-A-4-293495 correspondingto EP-A-504823 in which productivity of HSA is improved by adding fattyacids to a medium.

Isolation and recovery of HSA may be carried out using known processesdisclosed in the aforementioned references, or in accordance with aprocess disclosed in JP-A-3-103188 corresponding to EP-A-420007 in whichproteases are inactivated by heat treatment or a coloration inhibitionprocess disclosed in JP-A-4-54198 corresponding to U.S. Pat. No.5,132,404 or EP-A-464590 in which HSA is separated from coloringsubstances using at least one adsorbent selected from the groupconsisting of anion exchangers, hydrophobic carriers and activatedcharcoal.

The medium for culturing a transformed host may be prepared by addingfatty acids having 10 to 26 carbon atoms, or salts thereof, to a knownmedium, and culturing the transformant under known conditions. Themedium may be either synthetic or natural, but preferably a liquidmedium. For example, a suitable synthetic medium may be composed of:carbon sources, such as various saccharides and the like; nitrogensources, such as urea, ammonium salts, nitrates and the like; tracenutrients, such as various vitamins, nucleotides and the like; andinorganic salts, such as of Mg, Ca, Fe, Na, K, Mn, Co, Cu and the like.An illustrative example of such a medium is YNB liquid medium whichconsists of 0.7% Yeast Nitrogen Base (Difco) and 2% glucose. Anillustrative example of a useful natural medium is YPD liquid mediumwhich consists of 1% Yeast Extract (Difco), 2% Bacto Peptone (Difco) and2% glucose. The medium pH may be neutral, weakly basic or weakly acidic.In the case of a methanol assimilating host, the medium may be furthersupplemented with methanol in an amount of approximately from 0.01 to5%.

Culturing of a host may be carried out preferably at 15 to 43° C. (20 to30° C. for yeast strains, 20 to 37° C. for bacterial strains) for 1 to1,000 hours, by means of static or shaking culturing or batch,semi-batch or continuous culturing under agitation and aeration.

In that instance, it is desirable to prepare a seed culture prior to thebatch culturing. The seed culturing may be carried out using theaforementioned YNB liquid medium or YPD liquid medium, preferably at 30°C. (yeast) or 37° C. (bacterium) and for 10 to 100 hours.

After completion of the culturing, HSA is recovered from the resultingculture medium or cells in the usual way.

(2) Purification of HSA

(i) Ultrafiltration

High molecular weight substances other than HSA, as well as lowmolecular weight substances, are separated and removed from a culturesupernatant obtained after separation of HSA-producing host cells, usingultrafiltration techniques.

High molecular weight substances are removed using an ultrafiltrationmembrane having a molecular weight exclusive limit of approximately from100,000 to 500,000, preferably around 300,000, and low molecular weightsubstances are removed using another ultrafiltration membrane having amolecular weight exclusive limit of approximately from 1,000 to 50,000,preferably from about 10,000 to about 30,000.

Separation of remaining HSA-producing host cells is effectedsimultaneously at the time of the removal of high molecular weightsubstances, and concentration of the liquid is effected at the time ofthe removal of low molecular weight substances.

(ii) Heat treatment

The concentrated solution obtained in the above step (i) is subjected toheat treatment at 50 to 70° C. for approximately 30 minutes to 5 hours,preferably at 60° C. for approximately 1 to 3 hours.

Preferably, the heating is conducted in the presence of a stabilizingagent. Preferred examples of the stabilizer include acetyltryptophan andan organic carboxylic acid having 6 to 18 carbon atoms, or a saltthereof. The stabilizers may be used in combination. Acetyltryptophanmay be used in an amount of approximately from 1 to 100 mM. Illustrativeexamples of the organic carboxylic acid having 6 to 18 carbon atomsinclude caproic acid (6 carbon atoms), caprylic acid (8 carbon atoms),capric acid (10 carbon atoms), lauric acid (12 carbon atoms), palmiticacid (16 carbon atoms), oleic acid (18 carbon atoms) and the like.Illustrative examples of the salts include alkali metal salts such assodium salt, potassium salt and the like, and alkaline earth metalsalts, such as calcium salt and the like. The organic carboxylic acidhaving 6 to 18 carbon atoms or a salt thereof may be used in an amountof approximately from 1 to 100 mM.

In the heat treatment step, color development caused by the heating canbe prevented by the addition of a thiol compound (for example,mercaptoethanol, cysteine, reduced glutathione or the like) in an amountof approximately from 1 to 100 mM, preferably from 5 to 30 mM, andaminoguanidine in an amount of from 10 to 1,000 mM. A part of this stephas already been disclosed in JP-A-3-103188.

(iii) Acid treatment

The heat-treated solution of the above step (ii) is adjusted to a pH ofapproximately 3 to 5, preferably 4 to 4.6, with acid and then allowed tostand for a period of approximately 1 to 12 hours. Examples of the acidinclude acetic acid, hydrochloric acid, phophoric acid, sulfuric acidand the like.

(iv) Ultrafiltration

In this step, polymerized high molecular weight contaminants are removedby ultrafiltration. High molecular weight substances are removed usingan ultrafiltration membrane having a molecular weight exclusive limit ofapproximately from 100,000 to 500,000, preferably around 300,000. Ifnecessary, buffer exchange may be carried out for use in the followingcation exchanger treatment, using an ultrafiltration membrane having amolecular weight exclusive limit of approximately from 1,000 to 50,000,preferably from about 10,000 to about 30,000.

(v) Cation exchanger treatment

Usable as cation exchangers are those having sulfo groups, carboxylgroups and the like. Illustrative examples of the sulfo group-containingcation exchangers include sulfoagarose (trade name, S-Sepharose®,available from Pharmacia), sulfopropyl-dextran (trade name,SP-Sephadex®, available from Pharmacia), sulfopropyl-polyvinyl (tradename, SP-Toyopearl®, available from Tosoh Corp.) and the like.Illustrative examples of the carboxyl group-containing cation exchangersinclude carboxymethyl-dextran (trade name, CM-Sephadex®, available fromPharmacia; and trade name, CM-Cellulofine®, available from SeikagakuCorp.) and the like.

The exchanger can be equilibrated with an appropriate buffer such asacetate buffer having a pH of approximately 3 to 5, preferably 4 to 4.6and containing a salt such as sodium chloride in a concentration ofapproximately 0.01 to 0.2 M, preferably 0.05 to 0.1 M. The same buffercan be used for contacting and washing treatments. Elution may beeffected with an appropriate buffer such as phosphate buffer having a pHof generally 8 to 10, preferably 8.5 to 9.5 and containing a salt suchas sodium chloride in a concentration of generally 0.2 to 0.5 M,preferably 0.3 to 0.4 M.

(vi) First hydrophobic chromatography

Carriers for use in hydrophobic chromatography include those containingan alkyl group (butyl group, octyl group, octyldecyl group and thelike), each group having 4 to 18 carbon atoms, and those containing aphenyl group. Illustrative examples of the butyl group-containingcarriers include butyl-agarose, butyl-polyvinyl (trade name, ButylToyopearl®, available from Tosoh Corp.) and the like, those of the octylgroup-containing and octyldecyl group-containing carriers includeoctyl-agarose and octyldecyl-agarose, respectively, and those of thephenyl group-containing carrier include phenyl-cellulose (trade name,Phenyl Cellulofine®, available from Seikagaku Corp.) and the like.

In this step, HSA can be recovered from non-adsorbed fractions. In thatcase, contacting may be effected using an appropriate buffer such asphosphate buffer having a pH of approximately 6 to 8, preferably 6.5 to7 and containing a salt such as sodium chloride in a concentration ofapproximately 0.01 to 0.5 M, preferably 0.05 to 0.2 M.

HSA also can be recovered by elution after adsorption to theaforementioned carrier. In that case, contacting and washing may becarried out using an appropriate buffer such as phosphate buffer havinga pH of approximately 6 to 8, preferably from 6.5 to 7 and containing asalt such as sodium chloride in a concentration of approximately 1 to 3M, preferably 1.5 to 2 M. The elution may be effected with anappropriate buffer such as phosphate buffer having a pH of approximately6 to 8, preferably 6.5 to 7 and containing a salt such as sodiumchloride in a concentration of approximately 0.01 to 0.5 M, preferably0.05 to 0.2 M.

(vii) Anion exchanger treatment

Examples of anion exchangers include those containing thediethylaminoethyl (DEAE) group, those containing a quaternary aminoethyl(QAE) group and the like. Illustrative examples of the DEAEgroup-containing anion exchangers include DEAE-agarose (trade name,DEAE-Sepharose®, available from Pharmacia), DEAE-dextran (trade name,DEAE-Sephadex®, available from Pharmacia), DEAE-polyvinyl (trade name,DEAE-Toyopearl®, available from Tosoh Corp.) and the like. Illustrativeexamples of the QAE group-containing anion exchangers includeQAE-agarose (trade name, Q-Sepharose®, available from Pharmacia),QAE-polyvinyl (trade name, QAE-Toyopearl®, available from Tosoh Corp.)and the like.

In this step, HSA can be recovered from non-adsorbed fractions. In thatcase, contacting may be effected using an appropriate buffer such asphosphate buffer having a pH of approximately 6 to 8, preferably 6.5 to7 and a salt concentration of approximately 0.01 to 0.1 M.

HSA also can be recovered by elution after adsorption to theaforementioned carrier. In that case, contacting and washing may becarried out using the same buffer as described above except forcontaining a salt such as sodium chloride in a concentration of 0.001 to0.05 M and elution may be carried out with the same buffer but having asalt concentration of 0.05 to 1 M.

(viii) Salting-out

In this step, HSA is precipitated specifically by adding a saltcomponent to the sample solution to a final salt concentration ofapproximately 0.1 to 3 M, preferably 0.5 to 1.5 M and then adjusting theresulting solution to a pH of approximately 3 to 5, preferably 3.5 to4.5. Impurities in the supernatant fluid are removed by separating theHSA precipitate.

The thus precipitated HSA is dissolved in an appropriate buffersolution. Though not particularly limited, usable as salt components toadjust ionic strength are sodium chloride, potassium chloride, ammoniumsulfate, sodium (or potassium) thiocyanate, sodium sulfate and the like.Also, though not particularly limited, separation of the precipitatedHSA from the supernatant fluid may be effected preferably bycentrifugation, press separation, cross-flow membrane separation and thelike.

This step may be carried out preferably after the anion exchangertreatment step (vii), but may also be interposed between the cationexchanger treatment step (v) and the hydrophobic chromatographytreatment step (vi) or between the hydrophobic chromatography treatmentstep (vi) and the anion exchanger treatment step (vii).

(ix) Chelate resin treatment

The above purification steps may further contain a step of allowing HSAto contact with a chelate resin which has a specified ligand moiety.This step may be carried out preferably after the anion exchangertreatment or salting-out precipitation treatment, whichever is thelater.

Preferably, the carrier moiety of the chelate resin may have hydrophobicnature. Examples of such a type of carrier moiety include a copolymer ofstyrene and divinylbenzene, a copolymer of acrylic acid and methacrylicacid and the like.

Examples of the ligand moiety include a thiourea group, as well as apolyamine group (including a polyalkylene polyamine group such aspolyethylene polyamine or the like) which contains, in one molecule, aplurality of sub-groups consisting of a polyol group such as anN-methylglucamine group, an imino group, an amino group, anethyleneimino group and the like. Illustrative examples of preferredcommercially available chelate resins having the above-described carrierand ligand moieties, include DIAION CRB02® (ligand moiety,N-methylglucamine group, available from Mitsubishi Kassi Corp.), DIAIONCR20® (ligand moiety, --NH(CH₂ CH₂ NH)_(n) H, available from MitsubishiKasei Corp.), LEWATIT TP214® (ligand moiety, --NHCSNH₂, available fromBayer) and AMBERLITE CG4000®, all of which have a copolymer of styreneand divinylbenzene as the carrier moiety.

Preferred conditions for the chelate resin treatment are as follows.

pH: acidic or neutral (pH 3 to 9, preferably 4 to 7), period: at least 1hour, preferably 6 hours or more, ionic strength: 50 mmho or less,preferably 1 to 10 mmho,

mixing ratio: 0.1 to 100 g, preferably 1 to 10 g, of the resin based on250 mg of HSA (wet basis).

In the process, contaminated coloring substances derived from the rawmaterial or the host are adsorbed to the chelate resin, thus enablingreduction of coloration of HSA.

The steps (v), (vi), (vii) and (ix) may be carried out using a column orin a batchwise operation, with the use of a column being preferred.

Salting-out step (viii) can also be carried out after chelate resintreatment stop (ix) and prior to the below-described second hydrophobicchromatography step (x).

(x) Second hydrophobic chromatography

Free nonantigenic contaminants detectable by the phenol-sulfuric acidmethod are not fully removed from the HSA obtained through theabove-described steps (i) to (vii) and (ix).

The HSA obtained through the above-described treatments is allowed tocontact with a carrier for hydrophobic chromatography at a pH of 2 to 5,preferably 3 to 4 and a salt concentration of 0.4 to 1 M, preferably 0.4to 0.7 M. The elution can be effected at a pH of 6 to 8, preferably 6.5to 7 and a salt concentration of 0.01 to 0.3 M, preferably 0.05 to 0.2M. The above-described step (vi) may be replaced with this hydrophobicchromatography step. Thus, HSA which does not contain free nonantigeniccontaminants detectable by the phenol-sulfuric acid method can berecovered.

The term "phenol-sulfuric acid treatment" used herein means one ofcolorimetric determination of carbohydrates which comprises adding aphenol solution to a sample carbohydrate solution, adding concentratedsulfuric acid thereto, shaking the mixture to allow a furfuralderivative derived from the carbohydrate utilizing heat of solution toreact with phenol and colorimetrically determining the resulting coloredreaction product. The free nonantigenic contaminants detectable by thephenol-sulfuric acid method include neutral carbohydrates such aspentose and hexose, monocarbohydrate glycoside such as oligosaccharides,complex carbohydrates and uronic acid, methylated carbohydrate and thelike. These contaminants do not cause antigen-antibody reaction withantibodies against producer host-derived substances.

Carriers for use in the second hydrophobic chromatography are the sameas those used in the first hydrophobic chromatography.

HSA which does not contain free nonantigenic contaminants detectable bythe phenol-sulfuric acid method can be obtained by a treatment with aConA-immobilized carrier such as ConA-Sepharose (Pharmacia) and the likein place of this hydrophobic chromatography treatment. The ConAtreatment can be carried out by contacting the HSA fraction with aConA-immobilized carrier either in a batchwise or column operation at apH of 6 to 8 and a salt concentration of 0.01 to 0.1 M and recoveringnon-adsorbed fractions.

(xi) Treatment with boric acid or a salt thereof

Instead of the above treatment (x), the HSA obtained through theabove-described steps (i) to (vii) and (ix) can be treated with boricacid or a salt thereof to remove antigenic producer host-derivedcontaminants and pyrogen as well as free nonantigenic contaminantsdetectable by the phenol-sulfuric acid method.

Examples of the boric acid include orthoboric acid, metaboric acid,tetraboric acid and the like. The salts thereof include alkali metalsalts such as sodium salt and potassium salt, alkaline earth metal saltssuch as calcium salt, and the like. Calcium tetraborate is preferablyused. Boric acid or a salt thereof is added to a final concentration ofabout 0.01 to 1 M, preferably about 0.05 to 0.2 M. This treatment can becarried out at a pH of about 8 to 11, preferably about 9 to 10 for about1 to 10 hours. This treatment is preferably effected at a low electricconductivity, for example, 1 mS or less. The HSA concentration ispreferably low, for example, 5% or less, more preferably about 0.1 to3%.

After the treatment with boric acid or a salt thereof, the precipitateformed are removed by, for example, centrifugation or filtration and thesupernatant is recovered, concentrated and desalted.

(xii) Ultrafiltration

The HSA obtained after the above step (xi) is preferably subjected toultrafiltration using an ultrafiltration membrane having a molecularweight exclusive limit of about 100,000.

HSA thus purified through the above steps is substantially free fromcoloring, which means a coloring degree of the purified HSA rangesapproximately from 0.001 to 0.005 in terms of an A₅₀₀ nm /A₂₈₀ nm ratioin a 25% HSA solution. The term "a 25% HSA solution" used herein means asolution of 25% protein which may contain contaminated proteins in anamount lower than the detection limit, namely 0.1 ng/ml, other thanpurified HSA. Coloration of HSA is reduced to a level of from 1/2 to1/10 by the chelate resin treatment of the instant invention.Especially, absorbance at around 500 nm, namely reddish coloration, isreduced to a level of from 1/3 to 1/10.

In addition, fatty acids which adsorb or bind to HSA, derived from themedium or the host or secreted by the host, can be removed by thechelate resin treatment.

Amounts of fatty acids adsorbed to HSA which has been purified throughthe above steps (i) to (vii) and the additional salting-out step arereduced to a level of 1/10 or below, preferably 1/100 or below, by thechelate resin treatment.

Amounts of fatty acids adsorbed to HSA can be measured in accordancewith a generally used means such as Duncombe's extraction method (Clin.Chim. Acta., 9, 122-125 (1964)) or acyl-CoA synthetase (ACS)-acyl-CoAoxidase (ACOD) method in which ACS and ACOD are used.

The Duncombe's extraction method comprises, in principle, convertingfatty acids into copper salts using a copper reagent, extracting withchloroform and then subjecting the extract to color development withBathocuproin. The method can be carried out easily using a kit such asNEFA-Test Wako® (Wako Pure Chemical Industries, Ltd.) which containsBathocuproin.

On the other hand, the ACS-ACOD method comprises, in principle, reactingfatty acids with acyl-CoA synthetase and acyl-CoA oxidase to generate H₂O₂ and exposing the thus formed H₂ O₂ by a color development schemeusing oxidation-condensation reaction of a chromogenic substance in thepresence of peroxidase. That method also can be carried out easily usinga measuring kit such as NEFAC-Test Wako® (Wako Pure Chemical Industries,Ltd.).

Substances to be removed by the defatting method of the presentinvention are fatty acids and esters thereof which are derived from rawmaterials for HSA production, such as that derived from blood, a medium,a host or those secreted by the host.

Examples of fatty acids to be removed include saturated fatty acidshaving 8 to 20 carbon atoms, such as palmitic acid, stearic acid and thelike, and unsaturated fatty acids having 16 to 20 carbon atoms, such asoleic acid, linoleic acid, arachidonic acid and the like.

Since this step is effective for the removal of those fatty acids, itcan be applied to the defatting of HSA molecules to which those fattyacids are attached, independent of the origin of the HSA.

(3) Pharmaceutical preparation

The HSA thus obtained may be made into pharmaceutical preparations bygenerally known means such as 10 hours of heat sterilization at 60° C.,ultrafiltration, filter sterilization, dispensation, freeze-drying andthe like. An illustrative example of the pharmaceutical preparation ofthe present invention is a liquid preparation which contains HSA in anamount of 5 to 25%, has a pH of approximately 6.4 to 7.4 and has anosmotic pressure ratio of around 1.

The HSA-containing pharmaceutical preparation of the instant inventionmay contain stabilizers which include acetyltryptophan or a salt thereof(e.g., sodium salt) and sodium caprylate. Each stabilizer may be used inan amount of approximately 0.001 to 0.2 M, preferably 0.01 to 0.05 M ina 25% HSA solution. The sodium content may be 3.7 mg/ml or less. The HSApreparation may further contain pharmaceutically acceptable additivessuch as sodium chloride and the like.

In general, the stabilizers may be added prior to the aforementionedpreparation steps such as 10 hours of heat sterilization at 60° C.,ultrafiltration, filter sterilization, dispensation, freeze-drying andthe like. Therefore, not only preservation stability of HSA but also itsstability during the preparation process of the pharmaceuticalpreparation of the instant invention can be improved.

The HSA-containing pharmaceutical preparation thus obtained can be usedclinically as injections in the same manner as the case of the prior artplasma-derived HSA preparations. For example, it may be used for thepurpose of rapidly increasing blood volume, mainly at the time of shock,supplementing circulation blood volume, improving hypoproteinemia ormaintaining collagen osmotic pressure. More illustratively, theHSA-containing pharmaceutical preparation of the present invention canbe used effectively for the treatment of hypoalbuminemia caused by theloss of albumin (burn injury, nephrotic syndrome or the like) or by thereduction of albumin synthesizing ability (hepatic cirrhosis or thelike), as well as for the treatment of hemorrhagic shock and the like.

The pharmaceutical preparation may be administered gradually byintravenous injection or intravenous drip infusion, with a dose ofgenerally from 20 to 50 ml as a 25% HSA solution (5 to 12.5 g as HSA)per one administration for an adult. The dose may be changed optionallydepending on the age, symptoms, weight and the like of the patient.Properties of the purified recombinant HSA.

(4) Purified HSA

The HSA of the instant invention is a homogeneous substance having amolecular weight of about 67,000 and an isoelectric point of 4.6 to 5.0.The HSA consists of a monomer and contains substantially no dimers,polymers or decomposed products. In fact, the total content of dimers,polymers and hydrolyzed products is approximately 0.01% or less.

Also, the HSA of the instant invention contains substantially noproducer host-derived contaminants, such as protein, polysaccharide andthe like, which means contaminants having antigenecity detectable byimmunoassay such as EIA, RIA, PHA and so forth. Thus, the HSA of theinstant invention contains substantially no host-derived contaminantshaving antigenecity detectable by immunoassay. In the case of a 25% HSAsolution, the protein content may be 1 ng/ml or below, preferably 0.1ng/ml or below, and the polysaccharide content may be 10 ng/ml or below,preferably 1 ng/ml or below. In that case, the purity of the HSA iscalculated to be 99.999999% or more, preferably 99.9999999% or more.

The degree of coloring of the 25% HSA solution may be in the range offrom 0.01 to 0.05 in terms of an A₃₅₀ /A₂₈₀ ratio, from 0.001 to 0.02 asan A₄₅₀ /A₂₈₀ ratio and from 0.001 to 0.005 as an A₅₀₀ /A₂₈₀ ratio.

Further, the amount of fatty acids linked to the HSA may be one moleculeor less, preferably 0.1 molecule or less, per one HSA molecule.

Furthermore, the content of free nonantigenic contaminants detectable bythe phenol-sulfuric acid method is 1 μg or less, that of antigenicproducer host-related contaminants is 0.1 ng or less and that of pyrogenis 0.1 EU or less.

According to the instant invention, recombinant HSA can be purifiedefficiently. In addition, the instant invention can providesubstantially pure recombinant HSA which does not contain producerhost-related substances and other contaminants, and is sufficiently freefrom coloration.

The following examples are provided to further illustrate the instantinvention. It is to be understood, however, that the examples are not tolimit the scope of the present invention.

REFERENCE EXAMPLE 1

(1) Used strain, Pichia pastoris GCP101

A strain of Pichia pastoris, PC4130, obtained in accordance with theprocess disclosed in JP-A-2-104290, was made by digesting a plasmidpPGP1, containing a transcription unit which is constructed so as toexpress HSA under the control of an AOX1 promoter, with NcoI and thensubstituting the resulting NotI-digested fragment for the AOX1 generegion of a Pichia pastoris strain GTS115 (his4). The strain does notgrow well in a medium containing methanol as the carbon source (Mut⁻strain) because of the deletion of the AOX1 gene.

The strain PC4130 was inoculated into 3 ml of YPD medium (1% yeastextract, 2% Bacto Peptone and 2% glucose). After 24 hours of culturing,the cells were inoculated into 50 ml of YPD medium so that the celldensity should be adjusted to initial turbidity with an OD₅₄₀ of 0.1.After 3 days of culturing at 30° C., the resulting cells again wereinoculated into 50 ml of YPD medium at an initial cell turbidity of 0.1at OD₅₄₀. Thereafter, subculturing was repeated every 3 days in the samemanner. After each subculturing, cells were diluted with sterile waterand poured onto a 2% MeOH-YNBw/oa.a. plate (0.7% Yeast Nitrogen Basewithout Amino Acids, 2% methanol and 1.5% agar powder) in an inoculumsize of 10⁷ cells/plate, followed by 5 days of culturing at 30° C. tojudge the present/absence of colonies. Twenty colonies were found on the2% MeOH-YNBw/oa.a. plate after 12 days of the successive subculturing.Mut⁻ strains can hardly grow on the 2% MeOH-YNBw/oa.a. medium while Mut⁺strains can grow well. That is, advent of a colony means that the strainacquired the capacity of increased methanol assimilation and thus a Mut⁺strain was obtained. One of the thus obtained colonies was dilutedappropriately with sterile water and spread onto a 2% MeOH-YNBw/oa.a.plate to isolate single colonies. One of the resulting single colonieswas named GCP101.

(2) Culturing of the strain

(First seed culture)

A 1 ml portion of the strain which had been frozen in glycerol wasinoculated into a 1,000 ml baffled Erlenmeyer flask containing 200 ml ofYPD medium (see Table 1) and cultured at 30° C. for 24 hours withshaking.

                  TABLE 1                                                         ______________________________________                                        Composition of YPD medium                                                     Components    Concentration (g/L)                                             ______________________________________                                        Yeast extract 10                                                              Peptone       20                                                              Glucose       20                                                              ______________________________________                                    

(Second seed culture)

The first seed culture broth was inoculated into a 10 liter-jarfermentor containing 5 liters of YPD medium, and the second seedculturing was carried out at 30° C. for 24 hours with agitation and atan aeration rate of 5 liters per minutes. In the seed culturing, the pHof the medium was not controlled.

(Main culture)

The second seed culture broth was transferred into a 1,200liter-fermentor containing 250 liters of a batch culture medium (seeTable 2), and batch culturing was started with agitation and aerationunder an internal pressure of 0.5 kg/cm² and at a maximum aeration rateof 800 liter/min under atmospheric pressure. The agitation rate wascontrolled so that the level of dissolved oxygen in the medium wasmaintained at approximately 50 to 30% of the saturated dissolved oxygenconcentration. When the glycerol in the batch culture medium wasconsumed, addition of a feeding medium (see Table 3) was started.Feeding rate of the medium was controlled using a computer in such amanner that methanol did not accumulate in the culture medium, therebyeffecting a high density culturing. The medium pH was controlled at afixed level of 5.85 by the addition of 28% aqueous ammonia. Fordefoamation of the culture medium, an antifoam agent (Adecanol®,manufactured by Asahi Denka Kogyo K.K.) was added in an amount of 0.30ml/liter at the time of the commencement of the batch culture,thereafter adding a small amount when required.

                  TABLE 2                                                         ______________________________________                                        Composition of batch culture medium                                           Components     Amount per liter                                               ______________________________________                                        Glycerol              50.0   g                                                H.sub.3 PO.sub.4 (85%)                                                                              14.0   ml                                               CaSO.sub.4 · 2H.sub.2 O                                                                    0.6    g                                                K.sub.2 SO.sub.4      9.5    9                                                MgSO.sub.4 · 7H.sub.2 O                                                                    7.8    g                                                KOH                   2.6    g                                                Biotin solution *1    1.6    ml                                               YTM solution *2       4.4    ml                                               FeSO.sub.4 · 7H.sub.2 O                                                                    65.0   g                                                CuSO.sub.4 · 5H.sub.2 O                                                                    6.0    g                                                ZnSO.sub.4 · 7H.sub.2 O                                                                    20.0   g                                                MnSO.sub.4 · 4-5H.sub.2 O                                                                  3.0    g                                                H.sub.2 SO.sub.4      5.0    ml                                               ______________________________________                                         *1 Biotin solution: 0.2 g/l                                                   *2 YTM solution:                                                         

                  TABLE 3                                                         ______________________________________                                        Composition of feeding medium                                                        Components                                                                             Amount                                                        ______________________________________                                               YTM solution                                                                              2 ml                                                              Methanol 1,000 ml                                                      ______________________________________                                    

REFERENCE EXAMPLE 2

An HSA expression plasmid pMM042 was constructed using an AOX2 promoter(a mutant of the natural AOX2 promoter (YEAST, 5, 167-177, 1988; Mol.Cell. Biol., 9, 1316-1323, 1989), in which the 255th base upstream fromthe initiation codon of said promoter is changed from T to C) isolatedfrom the strain GCP101 obtained in Reference Example 1. The thusconstructed plasmid was introduced into Pichia pastoris GTS115 to obtaina transformant UHG42-3 (EP-A-506040). Thereafter, the thus obtainedtransformant was cultured in accordance with the procedure of ReferenceExample 1, thereby allowing the transformant to produce HSA.

EXAMPLE 1

[i] Isolation of culture supernatant--membrane fractions (I) and (II)

About an 800 liter portion of the culture broth obtained in ReferenceExample 1 was subjected to a filter press to isolate the culturesupernatant. The resulting supernatant subsequently was treated with anultrafiltration membrane having a molecular weight exclusive limit of300,000. Then, the resulting filtrate was concentrated to a volume ofabout 80 liters using an ultrafiltration membrane having a molecularweight exclusive limit of 30,000 [membrane fraction (I)].

Next, the membrane fraction (I) was heat-treated at 60° C. for 3 hoursin the presence of 5 mM of sodium caprylate, 10 mM of cysteine and 100mM of aminoguanidine at pH 7.5. The thus heat-treated solution wascooled down rapidly to about 15° C., adjusted to pH 4.5 and then treatedwith an ultrafiltration membrane having a molecular weight exclusivelimit of 300,000 [membrane fraction (II)]. Thereafter, using anultrafiltration membrane having a molecular weight exclusive limit of30,000, the buffer in the resulting albumin solution was replaced by a50 mM acetate buffer (pH 4.5) containing 50 mM of sodium chloride.

[ii] Cation exchanger treatment

The albumin solution obtained in the above step [i] was applied to acolumn packed with S-Sepharose® which had been equilibrated in advancewith a 50 mM acetate buffer (pH 4.5) containing 50 mM of sodiumchloride, the column was washed thoroughly with the same buffer and thenelution was carried out with a 0.1 M phosphate buffer (pH 9) containing0.3 M sodium chloride.

Polysaccharide content before and after the cation exchanger treatmentwas measured in accordance with the phenol-sulfuric acid method to findthat the polysaccharide content has been reduced by 1/20 by thistreatment.

[iii] Hydrophobic chromatography

The albumin solution eluted from the S-Sepharose® column was applied toa column packed with Phenyl Cellulofine® which has been equilibrated inadvance with a 50 mM phosphate buffer (pH 6.8) containing 0.15 M sodiumchloride. Since albumin does not adsorb to Phenyl Cellulofine® undersuch conditions, the albumin fractions which passed through the columnwere collected.

The albumin solution thus recovered was concentrated to a volume ofabout 50 liters using an ultrafiltration membrane having a molecularweight exclusive limit of 30,000, and at the same time, the buffer inthe albumin solution was replaced by a 50 mM phosphate buffer (pH 6.8).

[iv] Anion exchanger treatment

The albumin solution thus treated with hydrophobic chromatography,concentrated and buffer-exchanged in the above step [iii] was applied toa column packed with DEAE-Sepharose® which had been equilibrated inadvance with a 50 mM phosphate buffer (pH 6.8). Under such conditions,albumin was not adsorbed to the DEAE-Sepharose® but passed through thecolumn.

[v] Salting-out of HSA

To a 5% HSA solution was added sodium chloride to a final concentrationof 1 M. The resulting solution was adjusted to pH 3.5 with acetic acidto precipitate HSA, and the thus precipitated HSA was separated from thesupernatant fluid by centrifugation, thereby effecting removal ofimpurities. The albumin precipitate can be used as an injection bydissolving it in a liquid, concentrating and buffer-exchanging the thusdissolved solution using an ultrafiltration membrane having a molecularweight exclusive limit of 30,000, adding a stabilizing agent to the thustreated solution if necessary, and then subjecting the resultingsolution to filter sterilization.

[vi] HPLC analysis of purified HSA

The HSA preparation obtained after the purification step by hydrophobicchromatography was analyzed by means of HPLC gel filtration under thefollowing conditions.

(a) Column: TSK gel G3000SWxL® (Tosoh Corp.)

(b) Eluent: 0.3 M NaCl/50 mM phosphate buffer

(c) Detection: absorbance at 280 nm

As shown in FIG. 1, the purified HSA preparation was found as a singlepeak of HSA monomer.

[vii] Analysis of yeast-derived components

A culture supernatant of a yeast strain which does not produce albuminwas partially purified in accordance with the purification process ofthe instant invention, and separated into a protein fraction and apolysaccharide fraction. Rabbits were immunized with the proteinfraction or the polysaccharide fraction. Using an antiserum preparationobtained in that manner, detection of yeast-derived components in thepurified albumin solution was carried out by means of enzyme immunoassay(EIA).

Results of the detection of yeast-derived components in a sampleobtained after the salting-out step are shown in Table 4. The sample wassubjected to the measurement after adjusting the albumin concentrationto 250 mg/ml.

Total content of the yeast-derived proteins in the 250 mg/ml albuminsolution was found to be 1,360 ng/ml in the case of a sample obtainedafter treatment by the hydrophobic chromatography and was 5.4 ng/ml inan anion exchanger-treated sample, thus showing reduction of the totalcontent to a level of 1/250 by the latter purification step. Inaddition, the yeast-derived proteins were not detected in the purifiedsample obtained after the salting-out step at a detection limit.

[viii] Properties of the purified HSA

(1) Molecular weight

Measurement of molecular weight was carried out in accordance with theaforementioned HPLC gel filtration procedure. Molecular weight of thepurified HSA of the present invention was found to be about 67,000,which was almost the same as that of the plasma-derived HSA.

(2) Isoelectric point

Isoelectric point was measured by polyacrylamide gel electrophoresis,for example, using phastsystem® (Pharmacia). Isoelectric point of thepurified HSA of the present invention was found to be about 4.9, whichwas almost the same as that of plasma-derived HSA.

(3) Coloring degree

Absorbances at 280 nm, 350 nm and 450 nm were measured, and coloringdegrees were calculated as an A₃₅₀ /A₂₈₀ ratio and an A₄₅₀ /A₂₈₀ ratio.The A₃₅₀ /A₂₈₀ ratio and the A₄₅₀ /A₂₈₀ ratio of the purified HSA of theinstant invention were found to be about 0.02 and about 0.01,respectively, which were almost the same as those of plasma-derived HSA.

EXAMPLE 2

The culture broth obtained in Reference Example 2 was treated in thesame manner as the procedures described in Example 1. Properties of thethus purified HSA were almost the same as those of the purified HSAdisclosed in Example 1, in terms of molecular weight, isoelectric pointand coloring degree, as well as polysaccharide content, gel filtrationpattern and content of yeast-derived components.

EXAMPLE 3

A 1 ml portion of the 25% solution of purified HSA obtained in Example 1was mixed with 1 g of DIAION CRB02® (a chelate resin having astyrene-divinylbenzene copolymer as the carrier portion and anN-methylglucamine group as the ligand portion, manufactured byMitsubishi Kasei Corp.), and the resulting mixture was stirred for 24hours at room temperature at pH 6.8 and ionic strength of 5 mmho. Theresin then was washed with distilled water to recover the non-absorbedfraction. Thereafter, the thus purified HSA was assessed for theproperties as set forth in Example 1. Molecular weight, isoelectricpoint and gel filtration pattern of the thus obtained HSA were the sameas those of the HSA obtained in Example 1.

(1) Analysis of yeast-derived components

Results of the detection of yeast-derived components in the 25% HSAsolution by EIA, together with the results of Example 1, are shown inTable 4.

                  TABLE 4                                                         ______________________________________                                        Results of the detection of yeast-derived components                                         Protein     Polysaccharide                                                    content     content                                            Sample         (HSA purity)                                                                              (HSA purity)                                       ______________________________________                                        Anion exchanger treatment                                                                     5.4 ng/ml  40 ng/ml                                           (steps (i) to (iv) of                                                                        (99.99999784%)                                                 Ex. 1)                                                                        Salting-out treatment                                                                        <0.1 ng/ml   4 ng/ml                                           (steps (i) to (v) of                                                                         (99.99999996%)                                                                            (99.9999984%)                                      Ex. 1)                                                                        Steps (i) to (iv) of                                                                         Not measured                                                                               4 ng/ml                                           Ex. 1 and chelate resin    (99.9999984%)                                      treatment                                                                     Steps (i) to (v) of                                                                          <0.1 n.g/ml <1 ng/ml                                           Ex. 1 and chelete resin                                                                      (99.99999996%)                                                                            (99.9999996%)                                      treatment                                                                     ______________________________________                                    

(2) Coloring degree

Absorbances at 280 nm, 350 nm, 450 nm and 500 nm were measured, andcoloring degrees were calculated as an A₃₅₀ /A₂₈₀ ratio, an A₄₅₀ /A₂₈₀ratio and an A₅₀₀ /A₂₈₀ ratio. The A₃₅₀ /A₂₈₀ ratio, the A₄₅₀ /A₂₈₀ratio and the A₅₀₀ /A₂₈₀ ratio of the HSA of the instant invention werefound to be about 0.02, about 0.01 and about 0.002, respectively, whichwere almost the same as those of plasma-derived HSA.

(3) Linked fatty acid content

Measurement was carried out using NFEA-Test Wako® (Wako Pure ChemicalIndustries, Ltd.). The linked fatty acid content was about 1.6 moles(per mole of HSA) before the chelate resin treatment but was sharplyreduced by that treatment to 0.037 mole per mole of HSA.

EXAMPLE 4

The culture broth obtained in Reference Example 2 was treated in thesame manner as in Example 1. Properties of the 25% solution of thuspurified HSA were almost the same as those disclosed in Examples 1 and3, in terms of molecular weight, isoelectric point, coloring degree, gelfiltration pattern and content of yeast-derived components.

EXAMPLE 5

The 25% solution of the HSA obtained in Example 3 was checked forabsorbances at 280 nm and 500 nm. In that case, the coloring degree wascalculated as an A₅₀₀ /A₂₈₀ ratio. In addition, other HSA samples wereprepared by repeating the process of Example 3, except that DIAION CR20®(Mitsubishi Kasei Corp.) or LEWATIT TP214® (Bayer) was used instead ofDIAION CRB02®, and the absorbances were measured to calculate coloringdegrees. The results are shown in Table 5. As controls, several HSAsamples were prepared using carriers other than the chelate resin of thepresent invention, as well as other types of cation exchanger, anionexchanger and hydrophobic chromatography carrier, with the results alsoshown in Table 5.

                  TABLE 5                                                         ______________________________________                                        Coloring degree                                                               Resin                                                                         Carrier     Ligand        Trade name                                                                              Coloring*                                 ______________________________________                                        Invention                                                                     Styrene-divinyl                                                                           N-methylgluc- DIAION    0.2                                       benzene copolymer                                                                         amine group   CRB02 ®                                                                   (Mitsubishi)                                        Styrene-divinyl                                                                           --NH(CH.sub.2 CH.sub.2 NH).sub.n H                                                          DIAION    0.2                                       benzene copolymer         CR20 ®                                                                    (Mitsubishi)                                        Styrene-divinyl                                                                           --NHCSNH.sub.2                                                                              LEWATIT   0.2                                       benzene copolymer         TP214 ®                                                                   (Bayer)                                             Control                                                                       Styrene-divinyl                                                                           DEAE          DEAE-     0.5                                       benzene copolymer         Toyopearl ®                                                               (Tosoh)                                             Agarose     iminodiacetic Chelating-                                                                              0.8                                                   acid          Sepharose ®                                                               (Pharmacia)                                         Anion exchanger           DEAE      0.6                                                                 Sepharose ®                                                               (Pharmacia)                                         Hydrophobic               Phenyl    0.6                                       chromatography            Cellulo-                                                                      fine ®                                                                    (Pharmacia)                                         ______________________________________                                         Note                                                                          *: Coloring degree in an 0D.sub.500 /OD.sub.280 ratio which was defined a     1.0 in the case of the purified HSA obtained through Reference Example 1      and Example 1.                                                           

EXAMPLE 6 Measuring Wave Length

Absorbance of the 25% solution of HSA obtained in Example 3 was measuredat wave lengths of from 350 nm to 650 nm. Decreasing degrees ofabsorbance were found. The lowest ratio was found at a wave length of500 nm. The results are shown in Table 6.

                  TABLE 6                                                         ______________________________________                                        Decreasing degree of absorbance                                                          Decreasing ratio (%)                                               ______________________________________                                        A.sub.350 /A.sub.280                                                                       57                                                               A.sub.400 /A.sub.280                                                                       37                                                               A.sub.450 /A.sub.280                                                                       25                                                               A.sub.500 /A.sub.280                                                                       20                                                               A.sub.550 /A.sub.280                                                                       34                                                               A.sub.600 /A.sub.280                                                                       54                                                               A.sub.650 /A.sub.280                                                                       56                                                               ______________________________________                                         Starting material: 100%                                                  

EXAMPLE 7

The process of Example 3 was repeated except that DIAION CR20®(Mitsubishi Kasei Corp.) was used instead of the chelate resin DIAIONCRB02® in the treatment step using 1 ml of the 25% recombinant HSAsolution. The DIAION CR20® is a chelate resin having a --NH(CH₂ CH₂NH)_(n) H group as the ligand attached to a styrene-divinylbenzenecopolymer carrier. When the thus recovered HSA was checked for linkedfatty acids, similar results to those of the case of Example 6 wereobtained.

EXAMPLE 8

The process of Example 3 was repeated except that LEWATIT TP214® (Bayer)was used instead of the chelate resin DIAION CRB02® in the treatmentstep of 1 ml of the 25% recombinant HSA solution. LEWATIT TP214® is achelate resin having a --NHCSNH₂ group as a ligand attached to thestyrene-divinylbenzene copolymer carrier. When the thus recovered HSAwas checked for linked fatty acids, similar results to those of the caseof Example 6 were obtained.

EXAMPLE 9

Purity

Antibodies against the yeast components were prepared to detectimpurities of yeast components at a high level of sensitivity. Afterculturing non-albumin producing yeast, yeast components were partiallypurified from the culture supernatant and separated into a proteinfraction and a polysaccharide fraction. Each fraction was used toimmunize rabbits to obtain an antibody against the protein fraction andan antibody against the polysaccharide fraction. An EIA was developedusing these antibodies. In EIA system, the detection limit of proteinsand polysaccharides were 0.1 ng/ml and 1 ng/ml, respectively. Inpurified recombinant HSA obtained in Example 3, at a concentration of250 mg/ml, no yeast components were detected using this EIA system. Thatis, the contents of yeast-derived proteins and polysaccharides inpurified recombinant albumin at a concentration of 25% were less than0.1 ng/ml and less that 1 ng/ml, respectively. In other words, thepurity of recombinant albumin was greater than 99.999999%. ContaminatedDNA was assayed by the threshold method as described in Science, 240,1182 (1988). In purified recombinant albumin, no DNA was detected in a 2ml extract of a 25% albumin solution. Because the detection limit ofthis system was 4 pg/2 ml of 25% albumin, the amount of thecontaminating DNA was less than that value. Pyrogens were measured usinga reagent kit, Endospacy (Seikagaku Corp.). The pyrogen content was lessthan 0.1 EU/ml of 25% recombinant albumin, a sufficiently low level. Inthe pyrogen test using rabbits, there was no temperature rise up to adosage of 2.5 grams per kilogram.

Composition and Structure

To ascertain the composition and structure of recombinant albumin, aminoacid analysis was conducted and the CD spectrum was measured. The aminoacid composition and N and C terminal sequence of recombinant albuminwere identical to those of plasma albumin. Those results were consistentwith the sequence of the c-DNA. Peptide mapping of recombinant albuminwas carried out. Albumin was degradated by lysyl-endopeptiase, then eachpeptide was separated by reverse-phase HPLC. As a result of comparingthe peptide mapping patterns, the elution profile of recombinant albuminwas consistent with that of plasma albumin.

To examine the higher structure of recombinant albumin, the CD spectrumof albumin was measured. The CD spectrum of recombinant albumin wasidentical in the shape and magnitude to that of plasma albumin in theregion of 350 nm to 195 nm.

Biological Characterization

One of the most important biological functions of albumin is ligandbinding. Albumin binds various materials. The binding abilities ofalbumin to three typical materials were examined. Bilirubin was selectedto represent pigment. Warfarin was used to represent drugs and lauricacid to represent to fatty acids. The binding of those three materialswas analyzed using a Scatchard plot model. Binding constants and thenumber of binding sites of those three materials to recombinant albuminwere consistent with those of plasma albumin. Binding curves of lauricacid to albumin are shown in FIG. 2 as an example.

As shown in FIG. 2, binding curve of recombinant albumin was consistentwith plasma albumin.

The results that binding affinities of recombinant albumin with thoseligands were almost similar to those of plasma albumin indicate thebiological equivalency between both albumins.

Pre-Clinical Study

Preliminary data was collected during a pre-clinical study conducted inanimals. The half life of recombinant albumin in dog blood was almostidentical to that of plasma albumin (recombinant albumin; 6.3±0.5 day,plasma albumin; 6.0±0.7 day).

The following hemodynamic parameters were tested in dogs: bloodpressure; central venous pressure; pulmonary artery pressure; cardiacoutput; blood gas; respiration; and electrocardiogram. At doses of 0.5and 1.5 g/kg, the same results were obtained for recombinant albumin asfor plasma albumin.

The results of a pyrogen test using rabbits showed no temperature riseup to a dose of 2.5 g/kg.

An acute toxicity test of recombinant albumin demonstrated no toxicityin monkeys and rats up to a dosage of 12.5 g/kg.

EXAMPLE 10

In an appropriate volume of distilled water for injection were dissolved5 g of the yeast-derived HSA obtained in Example 1, 107.3 mg ofacetyltryptophan sodium salt and 66.5 mg of sodium caprylate to obtain20 ml of a pharmaceutical preparation containing 25% HSA. The resultingpharmaceutical preparation consisted of 25% HSA, 0.02 M acetyltryptophansodium salt and 0.02 M sodium caprylate. The sodium chloride content wasfound to be 3.7 mg/ml, the pH was 7.0 and the osmotic pressure was about1, as a ratio against physiologic saline.

EXAMPLE 11

Preservation stability of the HSA-containing pharmaceutical preparationobtained in Example 10 was examined. Determination of dimers (molecularweight distribution) was carried out by gel filtration analysis,coloring degree by absorbance analysis (A₃₅₀ /A₂₈₀) and polymers ordecomposed products by electrophoresis (SDS-PAGE). The results are shownin Tables 7 and 8. According to the instant invention, gelation of HSAdid not occur. The content of dimers increased gradually when maintainedat 40° C., but polymers or decomposed products were not found.

                  TABLE 7                                                         ______________________________________                                        Cryopreservation test at -20° C. for 8 weeks                           Sodium acetyl                                                                          Sodium           Before After Increasing                             tryptophan                                                                             caprylate                                                                              NaCl    preser-                                                                              preser-                                                                             ratio                                  (M)      (M)      (mg/ml) vation vation                                                                              (fold)                                 ______________________________________                                        (Coloring degree, A.sub.350 /A.sub.280)                                       0        0        0       0.0236 0.0277                                                                              1.17                                   0.02     0.02     3.7     0.0250 0.0261                                                                              1.04                                   (Dimer content, %)                                                            0        0        0       0.021  0.036                                        0.02     0.02     3.7     0.021  0.020                                        ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Accelerated preservation test at 40° C for 3 months                    (Dimer content, %)                                                            Sodium acetyl                                                                           Sodium            Before                                                                              After                                       tryptophan                                                                              caprylate                                                                              NaCl     preser-                                                                             preser-                                     (M)       (M)      (mg/ml)  vation                                                                              vation                                      ______________________________________                                        0         0        0        0.018  0.052                                                                              or more                               0.02      0.02     3.7      0.018  0.036                                      ______________________________________                                    

EXAMPLE 12

Sodium chloride was added to the purified HSA obtained in Example 3(without effecting salting-out [v] in Example 1) to a finalconcentration of 0.5 M. The resulting solution was adjusted to a pH of3.5 and applied to a column packed with Phenyl-Cellulofine. The columnwas washed with a 0.5 M sodium chloride solution (pH 3.5) and elutionwas carried out using 50 mM phosphate buffer (pH 6.8) containing 0.15 Msodium chloride.

EXAMPLE 13

The HSA concentration of the purified HSA obtained in Example 3(excluding the salting-out step [v] in Example 1) was adjusted to 2.5w/v % so that the electric conductivity became 1 mS or below. Calciumtetraborate was added to the resulting solution to a final concentrationof 100 mM and a pH value of the solution was adjusted to 9.5. Afterallowing the solution to stand for 10 hours, the precipitate formed wasremoved to recover the supernatant which was then concentrated anddesalted.

EXAMPLE 14

The purified HSA obtained in Example 13 was treated with aultrafiltration membrane having a molecular weight exclusive limit ofabout 100,000.

EXAMPLE 15

The HSA solution was treated in the same manner as in Example 13 exceptfor adding sodium tetraborate to a final concentration of 100 mM inplace of calcium tetraborate and adding calcium chloride to a finalconcentration of 100 mM.

EXAMPLE 16

HSA fractions (run Nos. 1 to 6 shown in Table 9) obtained through thesteps among the steps described in Example 1 shown in Table 9 wereexamined for the following items. The treatment with a ConA-immobilizedcarrier was carried out by applying the HSA fraction to a ConA-Sepharose(Pharmacia) column which had been equilibrated with a 50 mM phosphatebuffer (pH 6.8) and recovering non-adsorbed fractions.

(1) Determination of the content of free contaminants by thephenol-sulfuric acid method

The content of free contaminants in each HSA fraction was determined bythe phenol-sulfuric acid method in the conventional manner. Thus, eachHSA fraction was directly examined by the phenol-sulfuric acid method todetermine the total content of the contaminants (the sum of the freecontaminant content and the nonfree contaminant content). Separately,each HSA fraction was treated with ConA-Sepharose (Pharmacia) in thesame manner as described above and non-adsorbed fractions containing HSAwere subjected to the determination by the phenol-sulfuric acid methodto determine the content of nonfree contaminants. A difference obtainedby taking the latter from the former means the content of freecontaminants. A standard curve was prepared using mannnan as a standardmaterial. The results are shown in Table 9.

                  TABLE 9                                                         ______________________________________                                                                    Free Contami-                                     Run  Purification step      nant content                                      No.  [i]-[iv]                                                                              [v]    [vi] Example 12                                                                            ConÅ                                                                           (μg/250 mg of HSA)                   ______________________________________                                        1    do      --     --   --      --   1000                                    2    do      do     --   --      --   500                                     3    do      --     do   --      --   200                                     4    do      do     do   --      --    <1                                     5    do      do     --   do      --    <1                                     6    do      --     --   --      do    <1                                     ______________________________________                                         Note:                                                                         [i]-[iv] means the same step from isolation of culture supernatant to         anion exchanger treatment as in Example 1.                                    [v] means the same step of chelate resin treatment as in Example 3.           [vi] means the same step of saltingout in Example 1.                     

Example 12 means hydrophobic chromatography.

(2) Confirmation of nonantigenicity

The yeast-derived components in the purified HSA solution was determinedin the same manner as in Example 1 (vii). As a result, no yeast-derivedcomponent higher than the detectable limit of 0.1 ng/ml was detected inthe HSA fraction before and after the hydrophobic chromatographyaccording to the present invention.

EXAMPLE 17

The purified HSA (sample 1) obtained in Example 1 (excluding thesalting-out step [v]) was adjusted to the HSA concentration of 25 w/v %so that electric conductivity of the solution became 1 mS or loss.Calcium tetraborate was added thereto to a final concentration of 100 mMand a pH value was adjusted to 9.5. After allowing the mixture to standfor about 10 hours, the precipitate formed was removed to recover thesupernatant which was concentrated and desalted (sample 2). Theresulting HSA solution was treated with a ultrafiltration membranehaving a molecular weight exclusive limit of 100,000 (sample 3). Samples1, 2 and 3 were examined for the following items and the results areshown in Table 10.

(1) Recovery of HSA

The HSA content was determined by measuring the absorbance at 280 nm orby performing SDS-PAGE. As a result, recoveries of HSA in samples 2 and3 were both 95% or more.

(2) Determination of contaminants by EIA

The yeast-derived components were detected in the same manner as inExample 1 (vii). As a result, the content of the contaminants in sample1 was 10 ng/ml and no contaminant higher that the detectable limit of0.1 ng/ml was detected in samples 2 and 3.

(3) Determination of free contaminants by the phenol-sulfuric acidmethod

The content of free contaminants was determined in the same manner as inExample 16 (1).

(4) Determination of pyrogen

The content of pyrogen was determined using Endospecy (SeikagakuCorporation) in accordance with the manufacture's instruction attachedto the product.

                  TABLE 10                                                        ______________________________________                                                       Contaminant                                                                   content (per                                                                  250 ma of HSA)                                                                                 Phenol-                                                                       sulfuric                                                                            Pyrogen                                                  HSA            acid  (per 250                                Sample Purification                                                                            recov-         method                                                                              mg of                                   No.    step      ery     EIA    (free)                                                                              HSA)                                    ______________________________________                                        1      After decol-                                                                            --      10 ng  700 μg                                                                           2.9 EU                                         oration                                                                2      After boric                                                                             ≧95%                                                                           <0.1 ng                                                                              <1 μg                                                                            <0.1 EU                                        acid salt                                                                     treatment                                                              3      After ultra-                                                                            ≧95%                                                                           <0.1 ng                                                                              <1 μg                                                                            <0.1 EU                                        filtration                                                             ______________________________________                                    

While the instant invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to oneskilled in the art that various changes and modifications can be madetherein without departing from the spirit and scope thereof.

What is claimed is:
 1. A substantially pure recombinant human serumalbumin having a purity of 99.999999% or more, wherein a 25% solution ofsaid albumin contains contaminating proteins in an amount of 0.1 ng/mlor less and contaminating polysaccharides in an amount of 1 ng/ml orless and wherein a content of free nonantigenic contaminants detectableby a pheno-sulfuric acid method is 1 μg or less per 250 mg of saidalbumin.
 2. The recombinant human serum albumin according to claim 1,wherein a content of antigenic producer host-derived contaminates is 0.1ng or less and a content of pyrogen is 0.1 EU or less.
 3. Asubstantially pure recombinant human serum albumin having a purity of99.999999% or more, wherein a 25% solution of said albumin containscontaminating proteins in an amount of 0.1 ng/ml or less andcontaminating polysaccharides in an amount of 1 ng/ml or less.
 4. Apharmaceutical preparation comprising recombinant human serum albumin ofclaim 3, acetyltryptophan or a salt thereof and sodium caprylate.
 5. Thesubstantially pure recombinant human serum albumin according to claim 3,wherein the contaminating proteins and contaminating polysaccharides arederived from a host cell.
 6. The substantially pure recombinant humanserum albumin according to claim 5, wherein the host is a yeast strain.7. The substantially pure recombinant human serum albumin according toclaim 3, wherein the degree of coloring of a 25% solution of saidalbumin ranges from 0.01 to 0.05 in terms of an A₃₅₀ /A₂₈₀ ratio, from0.001 to 0.02 in terms of an A₄₅₀ /A₂₈₀ ratio and from 0.001 to 0.005 interms of an A₅₀₀ /A₂₈₀ ratio.
 8. The substantially pure recombinanthuman serum albumin according to claim 5, which is obtained by a processcomprising the step of exposing a recombinant human serumalbumin-containing fraction to a chelate resin and receivingnon-adsorbed fractions to yield albumin.
 9. The pharmaceuticalpreparation according to claim 4, wherein the albumin concentration is25% or 5%.
 10. A substantially pure recombinant human serum albuminhaving a purity of 99.999999% or more, wherein a 25% solution of saidalbumin contains contaminating proteins in an amount of 0.1 ng/ml orless and contaminating polysaccharides in an amount of 1 ng/ml or less,wherein the total content of dimers, polymers and hydrolyzed products is0.01% or less.